Three basic conditions must be met to achieve an efficient and gentle ionisation process in this technique:
- The ionisation must be resonant. The laser wavelength must be matched to achieve electronic or vibrational excitation of the molecule. Lasers with emission in the ultraviolet or remote infrared spectrum, respectively, are used for this purpose.
- The hot spot produced must be of very short duration, to avoid thermal decomposition of the molecule. Therefore, a laser with very short pulse duration (1 to 100 nanoseconds) is used.
- The sample must first be mixed with a matrix (usually a liquid), which sounds like small organic molecules that, with the evaporation of the solvent, forms crystals with the sample. In this way, the analyte molecules are isolated from each other and surrounded by a large number of matrix molecules, avoiding the likely association and formation of complexes between very high molecular weight analyte molecules that are difficult to desorb and analyse. In addition, the main function of the matrix is to absorb excess energy, resulting in extremely mild ionisation. With that energy absorption, the matrix converts it into excitation energy and H+ ions are transferred to the sample (ionisation), resulting in single-charged species.
This technique is combined with a TOF (Time of Flight) analyser.
Its principle is as follows: Once the sample is introduced into the source of the equipment with the matrix on a MALDI plate (made of steel), an intensive pulse of short wavelength laser shots is produced, causing the ionisation of the analyte. Once ionisation is achieved and all ions are confined in the source, an extraction voltage is applied, causing all ions to leave the source simultaneously. They then pass through an accelerating electrostatic field, acquiring a high kinetic energy that propels them in the direction of the flight tube towards the detector. The ions of higher m/z will fly at a slower speed than those of lower m/z, so the latter (smaller ions) will reach the detector first, and will be followed in time, successively, by the larger ions (provided they have the same charge).
The time taken to travel the length of the flight tube will be proportional to the mass or mass-to-charge ratio of the ions, and the detection system will be better able to distinguish, or differentiate, the different ion masses the greater the separation of the ions over time, the longer the flight tube (which due to its construction is considered a fixed value), or the smaller the energy dispersion of the ions formed at the source. A device called a reflectron is used to compensate for this dispersion and improve the resolution of the mass spectrum, which refocuses ions of the same mass (at about 5 KDa) onto the detector. In this way, MALDI equipment can work in two modes: linear mode (low resolution) for molecules larger than 5 KDa, and reflectron mode (high resolution) for molecules smaller than 5 KDa.
The matrices are used depending on the type of analyte to be measured. The matrices currently available, and which are the most common in MALDI, are the following:
|
Name |
Abbreviation |
Analyte |
|
α-Cyano-4-hydroxycinnamic acid |
HCCA |
Peptides Proteins (< 5 KDa) |
|
Sinapinic acid |
SA |
Proteins (10-150 KDa) Peptides (> 3 KDa) |
|
2,5-dihydroxybenzoic acid |
DHB |
Peptides Proteins Polymers Carbohydrates Lipids/Glycolipids |
|
1,8,9-trihydroxyanthracene (Dithranol) |
DIT |
Non-polar polymers |
|
Trans-2-[3-(4-(4-tert-Butylphenyl)-2-methyl-2-propenylidene] malonitrile |
DCTB |
Non-polar analytes |
For other matrices, please contact the service unit.
Sample type for MALDI
The MALDI technique is used for the analysis of peptides, carbohydrates, oligonucleotides, synthetic polymers, organometallic, organic and inorganic compounds, proteins, glycoproteins, polynucleotides, oligosaccharides and glycolipids.
Mass Spectrometry and Proteomics Unit
- Research Support Building (CACTUS)
- Rúa de Constantino Candeira, 1. Campus Vida , 15782Santiago de Compostela
- 881 816 242